ES2867553T3 - HCBI sequences as an early marker for the future development of CNS cancer and diseases and as a target for cancer treatment and prevention - Google Patents

Abstract

An HCBI polynucleic acid comprising: (a) a nucleotide sequence depicted in Figure 5 (SEQ ID. NO: 72); (b) a nucleotide sequence that is at least 90% identical to a nucleotide sequence of (a); (c) a nucleotide sequence that is complementary to a nucleotide sequence of (a) or (b); or (d) a nucleotide sequence that is redundant as a result of the degeneracy of the genetic code compared to any of the nucleotide sequences given above.

Description

DESCRIPTION

HCBI sequences as an early marker for the future development of CNS cancer and diseases and as a target for cancer treatment and prevention

The present invention relates to nucleotide sequences of HCBI (Healthy Cattle Blood Isolate) as well as to probes and primers comprising part of said nucleotide sequences and antibodies against polypeptides encoded by said nucleotide sequences. Finally, the present invention relates to the use of said compounds as an early marker for the future development of diseases such as cancer and CNS diseases and as a target for the treatment and prevention of cancer.

Several epidemiological analyzes carried out in the last decades indicate that the long-term consumption of "red" meat processed in different ways (including smoked meat and meat as a component of sausages) can be considered a risk factor for colon cancer ( World Cancer Report 2013, zur Hausen 2009, zur Hausen 2012). "Red" meat is considered to comprise beef, pork, sheep, lamb and goat, as opposed to "white" meat (poultry meat / fish).

Until now, carcinogenic chemicals produced during roasting, grilling, barbecuing, smoking, and air drying have been considered risk factors for cancer. However, the fact that the same substances are also produced in comparable concentrations during analogous forms of poultry meat / fish preparation was often ignored. Therefore, this does not support the assumption that these chemicals play a unique role in the development of colon cancer. Since, in addition, current epidemiological analyzes suggest that beef is the main risk factor, it has been postulated that an additional species-specific factor, presumably infectious, contributes to triggering this type of cancer (zur Hausen, 2012 ). The results of the correlation of the analyzes of the global spread of domesticated bovine species with the global incidence of colon cancer seem to suggest that the consumption of meat from bovine species from European / Asian cattle ( Bos taurus), but not from breeds Zebu, water buffalo or yak, may be important as a primary risk factor.

Therefore, the technical problem underlying the present invention is to identify specific nucleotide sequences that could be associated with diseases such as cancer or CNS diseases and therefore provide means for diagnosis and therapy.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims. During the experiments that resulted in the present invention, cattle sera were analyzed for infectious agents, based on the assumption that the presence in sera is also indicative of the presence of these agents in "red" meat. Sera from healthy cows were analyzed and five new viral nucleic acid components could be isolated. The DNA sequences and open reading frames of these components showed a recognizable relationship with the sequences already described for transmissible spongiform encephalopathies (TSE) for TSE diseases in sheep, cattle and humans.

TSE isolates have also been suspected of playing a role in cancer induction (Manuelidis, 2011), so it is reasonable to assume that the viral sequences described could be associated with the development of diseases such as cancer and diseases of the CNS. Brassard et al., Veterinary Microbiology, Vol. 126, No. 1-3, pp. 271-276 (2007) describe the molecular detection of bovine and porcine Torque teno virus in plasma and feces. Schuurman et al., Biologicals, Vol. 19, pp. 265-270 (1991) describe that bovine polyomavirus is a frequent contamination of calf serum. Leary et al., J. of General Virology, Vol. 80, pp. 2115-2120 (1999) describe improved systems for the detection of TT viruses. Fuchs et al., J. of Clinical Microbiology, Vol. 37, No. 8., pp. 2498-2507 (1999) describe the detection of bovine herpesvirus 1 in the blood of naturally infected cattle by the use of sensitive PCR that discriminates between wild-type viruses and viruses lacking the glycoprotein E. Sprardbrow et al., J. of Comparative Pathology, Vol. 97, No. 4, pp. 469-479 (1987) describe a connection between skin cancer and papillomaviruses in cattle. Giovanny et al., Open J. of Medical Microbiology, Vol. 3, pp. 84-90 (2013) describe that a bovine leukemia virus gene segment has been detected in human breast tissue.

Brief description of the drawings

Figure 1: Nucleotide sequence of HCBI1.225 and putative open reading frames (Comparative Figure) Also shown are alignments with nucleotide sequences found in databases and alignments of peptides derived from open reading frames of HCBI1.225 a the corresponding "Sphinx" amino acid sequences.

Figure 2: Nucleotide sequence of HCBI2.170 and putative open reading frames (Comparative Figure) Also shown are alignments with nucleotide sequences found in databases and alignments of peptides derived from open reading frames of HCBI2.170 a the corresponding "Sphinx" amino acid sequences.

Figure 3: Nucleotide sequence of HCBI3.108 and putative open reading frames (Comparative Figure) Also shown are alignments with nucleotide sequences found in databases and alignments of peptides derived from open reading frames of HCBI3.108 a the corresponding "Sphinx" amino acid sequences.

Figure 4: Nucleotide sequence of HCBI4.296 and putative open reading frames (Comparative Figure) Also shown are alignments with nucleotide sequences found in databases and alignments of peptides derived from open reading frames of HCBI4.296 a the corresponding "Sphinx" amino acid sequences.

Figure 5: Nucleotide sequence of HCBI5.173 and putative open reading frames

Also shown are alignments with the nucleotide sequences found in the databases and alignments of peptides derived from HCBI5.173 open reading frames to the corresponding "Sphinx" amino acid sequences.

Accordingly, the present invention relates to a HCBI polynucleic acid comprising:

(a) a nucleotide sequence depicted in Figure 5;

(b) a nucleotide sequence that is at least 90% identical to a nucleotide sequence of (a);

(c) a fragment of a nucleotide sequence of (a) or (b);

(d) a nucleotide sequence that is complementary to a nucleotide sequence of (a), (b), or (c); or (e) a nucleotide sequence that is redundant as a result of the degeneracy of the genetic code compared to any of the nucleotide sequences given above.

The term "polynucleic acid" refers to a single or double stranded nucleic acid sequence. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogs or modified nucleotides, or it may have been adapted for diagnostic or therapeutic purposes. A polynucleic acid can also comprise a double-stranded cDNA clone that can be used, for example, for cloning purposes.

The HCBI5.173 polynucleic acids of the invention can be prepared according to well known routine procedures, for example, (a) isolating complete DNA or RNA from a sample, (b) detecting the HCBI sequence by hybridization or PCR and (c) cloning the HCBI sequence into a vector.

Also included within the present invention are sequence variants of the polynucleic acid of the invention that contain deletions and / or insertions of one or more nucleotides, especially insertions or deletions of one or more codons, mainly at the ends of the oligonucleotides (either 3 'or 5') and showing at least 90%, 95% or 98% identity with said polynucleic acid sequences of the invention. Polynucleic acid sequences according to the present invention that are similar to the sequence shown in Figure 5 can be characterized and isolated according to any of the techniques known in the art, such as amplification by means of specific primers of sequence, hybridization with sequence-specific probes under more or less stringent conditions, determination of the sequence of the HCBI genetic information, etc.

The present invention also provides fragments of the nucleotide sequences of the present invention described above that are preferably capable of autonomously replicating as described in Example 1. An autonomously replicating nucleotide sequence comprises a nucleotide sequence of the gene replication or a fragment thereof that is capable of inducing autonomous replication. The replication protein represents an endonuclease that binds to single-stranded DNA that induces a single-stranded cut at or near the origin of replication (Wolds, 1997). The skilled person can obtain such fragments capable of inducing autonomous replication without excessive experimentation. Said fragments can have a length of at least 45, at least 55 or at least 65 nt.

One of skill in the art can easily determine which nucleic acid sequences are related to a nucleotide sequence of Figure 5 or which fragments are still capable of autonomously replicating by using standard assays.

The present invention also provides polynucleic acid sequences that are redundant as a result of the degeneracy of the genetic code compared to any of the nucleotide sequences given above. These variant polynucleic acid sequences will thus encode the same amino acid sequence as the polynucleic acids from which they are derived.

The HCBI5.173 polynucleic acid of the invention could be present as an extrachromosomal episome, could integrate into the host genome and / or could be linked to a DNA of a host cell.

The present invention also relates to an oligonucleotide primer comprising or consisting in part of a polynucleic acid as defined above, said primer being able to act as a primer to specifically sequence or specifically amplify the HCBI5.173 polynucleic acid of the invention.

The term "primer" refers to a sequence of single-stranded DNA oligonucleotides capable of acting as a The starting point for the synthesis of a primer extension product that is complementary to the nucleic acid strand to be copied. The length and sequence of the primer must be such as to allow the synthesis of the extension products to be primed. Preferably, the primer is about 5-50 nucleotides. The specific length and sequence will depend on the complexity of the DNA or RNA targets required, as well as the conditions of use of the primer, such as temperature and ionic strength.

The fact that the amplification primers do not have to exactly match the corresponding template sequence to ensure adequate amplification is widely documented in the literature. The amplification procedure used can be, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), transcription-based amplification system (TAS), strand displacement amplification (SDA) or Amplification by means of Qp replicase or any other suitable method to amplify nucleic acid molecules using primer extension. During amplification, the amplified products can conveniently be labeled using labeled primers or incorporating labeled nucleotides.

The labels can be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplification reaction is repeated between 20 and 70 times, advantageously between 25 and 45 times.

Any of a variety of sequencing reactions known in the art can be used to directly sequence viral genetic information and determine the orf by translating the sample sequence into the corresponding amino acid sequence. Exemplary sequencing reactions include those based on techniques developed by Sanger or Maxam and Gilbert. It is also contemplated that a variety of automated sequencing procedures may be used when performing subject assays, including mass spectrometric sequencing (see, for example: PCT publication WO 94/16101). It will be apparent to one of ordinary skill in the art that, for example, the occurrence of only two or three nucleic bases in the sequencing reaction must be determined.

Preferably, these primers are about 5 to 50 nucleotides in length, more preferably about 10 to about 25 nucleotides in length. The most preferred primers are those that are at least 13 bases in length.

The present invention also relates to an oligonucleotide probe comprising or consisting in part of a polynucleic acid of HCBI5.173 as defined above, said probe being capable of acting as a hybridization probe for the specific detection of a polynucleic acid of HCBI5.173 according to the invention.

The probe can be labeled or attached to a solid support.

The term "probe" refers to single-stranded sequence-specific oligonucleotides having a sequence that is complementary to the target sequence of a HCBI5.173 polynucleic acid to be detected.

Preferably, these probes are about 5 to 50 nucleotides in length, more preferably about 10 to about 25 nucleotides in length. The most preferred probes are those that are at least 13 bases in length.

The term "solid support" can refer to any substrate to which an oligonucleotide probe can be coupled, as long as it maintains its hybridization characteristics and as long as the background level of hybridization is kept low. Generally, the solid substrate will be a microtiter plate, a membrane (eg, nylon or nitrocellulose), or a microsphere (bead). Before application to the membrane or fixation, it may be desirable to modify the nucleic acid probe to facilitate fixation or improve the efficiency of hybridization. Such modifications can encompass homopolymer tailing, coupling with different reactive groups, such as aliphatic groups, NH2 groups, SH groups, carboxylic groups, or coupling with biotin or haptens.

The oligonucleotides according to the present invention, used as primers or probes can also contain or consist of nucleotide analogs such as phosphorothioates, alkyl phosphorates or peptide nucleic acids or can contain intercalating agents. These modifications will require adaptations with respect to the conditions under which the oligonucleotide must be used to obtain the required specificity and sensitivity. However, the final results will be essentially the same as those obtained with the unmodified oligonucleotides.

The introduction of these modifications can be advantageous to positively influence characteristics such as hybridization kinetics, reversibility of hybrid formation, biological stability of oligonucleotide molecules, etc.

The polynucleic acids of the invention can be comprised in a composition of any type. Said composition can be for diagnostic, therapeutic or prophylactic use.

The present invention also relates to a recombinant expression vector comprising a polynucleic acid of HCBI5.173 of the invention as defined above operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements, as well as to host cells containing said vector.

The term "vector" can comprise a plasmid, a cosmid, an artificial chromosome, a phage or a virus or a non-human transgenic animal. They may be particularly useful for the development of vaccines, recombinant HCBI5.173 molecules, BCG, or adenoviral vectors, as well as recombinant avipox viruses.

The term "recombinant expression" used in the context of the present invention refers to the fact that the polypeptides of the present invention are produced by recombinant expression methods, either in prokaryotes or lower or higher eukaryotes, as described in detail below continuation.

The term "host cell" refers to cells that can be, or have been, used as recipients of a recombinant vector or other transfer polynucleotide, and include the progeny of the original cell that has been transfected.

It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental or deliberate mutation or recombination.

The term "lower eukaryotic" refers to host cells such as yeast, fungi, and the like. Lower eukaryotes are generally (but not necessarily) single-celled. Preferred lower eukaryotes are yeasts, particularly species within Saccharomyces, Schizosaccharomyces, Kluiveromyces, Pichia (eg Pichia pastoris), Hansenula (eg Hansenula polymorph), Schwaniomyces, Schizosaccharomyces, Yarowia, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis, and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.

The term "higher eukaryotic" refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Currently preferred higher eukaryotic host cells are derived from Chinese hamster (eg CHO), monkey (eg COS and Vero cells), newborn hamster kidney (BHK), pig kidney (PK15) cells , rabbit kidney 13 (RK13), human osteosarcoma 143 B cell line, human HeLa cell line and human hepatoma cell lines such as Hep G2, 293TT cell line (Buck et al., 2004) and from insect cell lines (eg, Spodoptera frugiperda). Host cells can be provided in suspension or flask cultures, tissue cultures, organ cultures, and the like. Alternatively, the host cells can also be non-human transgenic animals.

The term "prokaryotes" refers to hosts such as E. coli, Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus subtilis, or Streptomyces. These hosts are also contemplated within the present invention.

The HCBI5.173 DNA segment encoding the desired sequence inserted into the vector sequence can be linked to a signal sequence. Said signal sequence may be from a source other than HCBI5.173, but particularly preferred constructs according to the present invention contain signal sequences that appear in the HCBI genome before the respective protein start points.

Higher eukaryotes can be transformed with vectors, or they can be infected with a recombinant virus, eg, a recombinant vaccinia virus. Techniques and vectors for inserting foreign DNA into vaccinia virus are well known in the art and use, for example, homologous recombination. A wide variety of viral promoter sequences are available in the art, possibly termination sequences and poly (A) addition sequences, possibly enhancer sequences, and possibly amplification sequences, all required for expression in mammals. Vaccinia is particularly preferred since vaccinia stops host cell protein expression. For vaccination of humans, the avipox and Modified Ankara viruses (AMV) are particularly useful vectors.

Insect expression transfer vectors derived from Autographa californica nuclear polyhedrosis baculovirus virus (AcNPV), which is a helper-independent viral expression vector, are also known. Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes. The person skilled in the art has different vectors, as well as procedures for the introduction of heterologous DNA into the desired baculovirus site for the expression of baculovirus. Various signals for post-translational modification recognized by insect cells are also known in the art.

The present invention also relates to a polypeptide having an amino acid sequence encoded by an HBCI polynucleic acid as defined above, or a part or analog thereof that is substantially similar and biologically equivalent.

The term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product.

Therefore, they are included within the definition of polypeptide, peptides, oligopeptides, and proteins. This term also does not refer to or exclude post-expression modifications of the polypeptide, eg, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, peptide nucleic acid (PNA), etc.), polypeptides with substituted bonds, as well as other modifications. known in the art, both natural and unnatural.

The polypeptides according to the present invention preferably contain at least 3, preferably 4 or 5 contiguous HCBI5.173 amino acids, preferably 6 or 7, however, at least 8 contiguous HCBI5.173 amino acids, at least 10 or at least 15 .

The polypeptides of the invention, and particularly the fragments, can be prepared by classical chemical synthesis. The synthesis can be carried out in homogeneous solution or in solid phase. Polypeptides according to the present invention can also be prepared by means of recombinant DNA techniques. The present invention also relates to a process for the production of a recombinant polypeptide as defined above, comprising: (a) transforming an appropriate cellular host with a recombinant vector, in which a polynucleic acid or a part thereof, as defined above, it has been inserted under the control of the appropriate regulatory elements, (b) culturing said transformed cell host under conditions that allow the expression of said insert, and (c) harvesting said polypeptide.

The present invention also relates to an antibody generated after immunization with at least one polypeptide as defined above, said antibody being specifically reactive with any of said polypeptides, and said antibody preferably being a monoclonal antibody. The term "antibody" preferably refers to antibodies consisting essentially of monoclonal antibodies combined with different epitopic specificities, as well as different monoclonal antibody preparations. Monoclonal antibodies are made from an antigen containing, for example, a polypeptide encoded by a HCBI5.173 polynucleic acid of the invention or a fragment thereof by procedures well known to those of skill in the art. As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is intended to include intact molecules, as well as antibody fragments (such as, for example, Fab and F (ab ') 2 fragments ) that are capable of specifically binding to protein. Fab and F (ab ') 2 fragments lack the Fc fragment of the intact antibody, are cleared more rapidly from the circulation, and may have less binding to non-specific tissues than an intact antibody. Therefore, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. In addition, antibodies useful for the purposes of the present invention include chimeric, single chain, and humanized antibodies.

Preferably, the antibody or antigen-binding fragment thereof carries a detectable label. The antibody / fragment may be detectably directly or indirectly labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Other suitable markers for binding the antibody will be known to, or will be able to determine, those of skill in the art using routine experimentation.

The present invention also relates to a diagnostic kit for use in determining the presence of a HCBI5.173 polynucleic acid or polypeptide of the invention, said kit comprising a primer, a probe and / or an antibody of the invention. .

The present invention also relates to a method for the detection of a polynucleic acid of HCBI5.173 according to the invention present in a biological sample, comprising: (a) extracting, optionally, the polynucleic acid from the sample, (b ) amplifying the polynucleic acid as described above with at least one primer as defined above, optionally a labeled primer, and (c) detecting the amplified polynucleic acids.

The term "polynucleic acid" can also refer to the analyte strand and corresponds to a single or double stranded polynucleic acid molecule.

The term "tagged" refers to the use of tagged nucleic acids. This may include the use of labeled nucleotides incorporated during the polymerase amplification step or of labeled primers, or by any other method known to those skilled in the art.

The present invention also relates to a method for the detection of a polynucleic acid of HCBI5.173 according to the invention present in a biological sample, comprising: (a) extracting, optionally, the polynucleic acid from the sample, (b ) hybridizing the polynucleic acid as described above with at least one probe as defined above and (c) detecting the hybridized polynucleic acids.

Hybridization and wash conditions are to be understood as stringent and are generally known in the art. However, depending on the hybridization solution (SSC, SSPE, etc.), these probes must hybridize at their appropriate temperature to achieve sufficient specificity.

According to the hybridization solution (SSC, SSPE, etc.), these probes must be rigorously hybridized to their appropriate temperature to achieve sufficient specificity. However, by slightly modifying the DNA probes, either by adding or removing one or a few nucleotides at their ends (either 3 'or 5'), or by substituting some non-essential nucleotides (i.e., non-essential nucleotides to discriminate between types ) by others (including modified nucleotides or inosine) these probes or variants thereof can be made to specifically hybridize under the same hybridization conditions (ie, the same temperature and the same hybridization solution). It may also be beneficial to change the amount (concentration) of the probe used to obtain more specific hybridization results. It should be noted in this context, that probes of the same length, regardless of their GC content, will specifically hybridize at approximately the same temperature in TMACI solutions.

Suitable assay procedures for the purposes of the present invention to detect hybrids formed between oligonucleotide probes and HCBI5.173 polynucleic acid sequences in a sample may comprise any of the assay formats known in the art, such as the format conventional dot blot, sandwich hybridization or reverse hybridization. For example, detection can be performed using a dot blot format, the unlabeled amplified sample is bound to a membrane, the membrane is incorporated with at least one labeled probe under suitable hybridization and wash conditions, and the presence of the attached probe.

An alternative and preferred procedure is a "reverse" dot blot format, in which the amplified sequence contains a tag. In this format, the unlabeled oligonucleotide probes are attached to a solid support and exposed to the labeled sample under appropriate post-wash and stringent hybridization conditions. It should be understood that any other assay procedure that relies on the formation of a hybrid between sample polynucleic acids and oligonucleotide probes may also be used in accordance with the present invention.

The present invention also relates to a method for detecting a polypeptide encoded by an HCBI5.173 polynucleic acid of the present invention or an antibody against said polypeptide present in a biological sample, comprising: (a) contacting the biological sample to detect the presence of said polypeptide or antibody as defined above, and (b) detect the immune complex formed between said antibody and said polypeptide.

Immunoassay procedures according to the present invention can utilize antigens from different domains of the new and unique polypeptide sequences of the present invention. It is within the scope of the invention to use, for example, individual or specific oligomeric antigens, dimeric antigens, as well as combinations of individual or specific oligomeric antigens. The HCBI antigen of the present invention can be used in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the HCBI conformational epitope should be avoided or adapted. A common feature of all of these assays is that the antigen is contacted with the component of the body suspected of containing HCBi antibodies under conditions that allow the antigen to bind to any such antibody present on the component. Such conditions will normally be physiological temperature, pH and ionic strength using excess antigen. Incubation of the antigen with the sample is followed by the detection of immune complexes comprised by the antigen.

The design of immunoassays is subject to great variation, and many formats are known in the art. The protocols can, for example, use solid supports or immunoprecipitation. Most assays involve the use of a labeled antibody or polypeptide; the labels can be, for example, enzymatic, fluorescent, chemiluminescent, radioactive or dye molecules. Assays that amplify the signals of the immune complex are also known; examples of which are assays using biotin and avidin or streptavidin, and labeled and enzyme-mediated immunoassays, such as ELISA assays.

The immunoassay can be in a heterogeneous or homogeneous format, and of a conventional or competitive type. In a heterogeneous format, the polypeptide is normally bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation. Examples of solid supports that can be used are nitrocellulose (for example, in membrane or microtiter well form), polyvinyl chloride (for example, in microtiter slides or wells), polystyrene latex (for example, in beads or plates microtiter), polyvinylidine fluoride (known as Immunolon), diazotized paper, nylon membranes, activated beads, and protein A beads. The solid support containing the antigenic polypeptides is normally washed after separation from the test sample and before detection of bound antibodies. Both conventional and competitive formats are known in the art.

In a heterogeneous format, the test sample is incubated with the antigen pool in solution. For example, it may be under conditions that precipitate any antigen-antibody complex that is formed. Both conventional and competitive formats for these assays are known in the art.

In a conventional format, the amount of HCBI antibodies in the antigen-antibody complexes is directly controlled. This can be accomplished by determining whether anti-xenogeneic (labeled) antibodies (eg, anti-human), which recognize an epitope on anti-HCBI antibodies, will bind due to complex formation. On In a competitive format, the amount of HCBI antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competitive ligand) in the complex.

The complexes formed comprising anti-HCBI antibodies (or in the case of competitive assays, the amount of competing antibodies) are detected by a number of known techniques, depending on the format. For example, unlabeled HCBI antibodies in the complex can be detected using an anti-xenogeneic Ig conjugate complexed with a tag (eg, an enzyme tag).

In an immunoprecipitation or agglutination assay format, the reaction between the HCBI antigens and the antibody forms a network that precipitates from solution or suspension and forms a visible layer or film of precipitate. If there is no anti-HCBI antibody in the test sample, a visible precipitate does not form.

There are currently three specific types of particle agglutination (PA) assays. These assays are used for the detection of antibodies against various antigens when they coat a support. One type of this assay is the hemagglutination assay that uses red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to RBCs. The addition of specific antigen / antibodies present on the body component, if any, causes the red blood cells coated with the purified antigen to agglutinate.

To eliminate possible non-specific reactions in the hemagglutination assay, two artificial carriers can be used in place of RBC in the AP. The most common of these are latex particles.

The selected solid phase can include glass or polymeric beads, nitrocellulose, microparticles, microwells of a reaction tray, test tubes, and magnetic beads. The signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element, and a chemiluminescent compound. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin, and avidin. Examples of binding members to enhancer compounds include biotin, anti-biotin, and avidin.

The above procedures are useful in assessing the risk of developing diseases such as cancer or an autoimmune disease due to the deleterious effects of the presence of a subgenomic HCBI polynucleotide sequence on its own or attached to a particular host gene or gene fragment within. cells of the patient and allows to take the appropriate measures.

Therefore, the present invention also relates to an antisense oligonucleotide or RNAi specific for the polynucleic acid of the HCBI5.173 virus of the invention.

Generation of suitable antisense oligonucleotides or RNAi includes determining a site or sites within the HCBI5.173 polynucleic acid for antisense interaction to occur so as to result in the desired effect, for example, inhibition of expression of the polypeptide. A preferred intragenic site is (a) the region spanning the translation initiation or termination codon of the open reading frame (ORF) of the gene or (b) a region of the mRNA that is a "loop" or "bulge", that is, it is not part of a secondary structure. Once one or more target sites have been identified, oligonucleotides are chosen that are sufficiently complementary to the target, that is, they hybridize well enough and with sufficient specificity, to produce the desired effect. In the context of the present invention, "hybridization" means hydrogen bonding, which may be inverted Watson-Crick, Hoogsteen or Hoogsteen hydrogen bonding, between complementary nucleoside bases or nucleotides. "Complementary" as used herein refers to the ability to precisely match two nucleotides. For example, if a nucleotide at a certain position in an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position in a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered complementary to each other in that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond to each other. Therefore, "specifically hybridizable" and "complementary" are expressions that are used to indicate a sufficient degree of complementarity or a precise pairing so that a stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound can specifically hybridize when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to prevent nonspecific binding. of the antisense compound to non-target sequences under conditions where specific binding is desired, that is, in the case of therapeutic treatment.

"Oligonucleotide" (particularly in the context of antisense compounds) refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of natural nucleobases, sugars, and covalent internucleoside bonds (backbones), as well as oligonucleotides that have unnatural portions that function in a similar manner. Such modified or substituted oligonucleotides are often preferred over natural forms due to desirable properties such as, for example, improved cell uptake, increased affinity for the nucleic acid target, and increased stability in the presence of nucleases. Although antisense oligonucleotides are a preferred form of the antisense compound, the present invention encompasses other oligomeric antisense compounds, including, but not limited to, oligonucleotide mimetics, as described below. Antisense compounds according to the present invention comprise from about 8 to about 50 nucleobases (ie, from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 15 to about 25 nucleobases.

Antisense compounds include ribozymes, external guide sequences (EGS), oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides that hybridize to the target nucleic acid and inhibit its expression. Antisense compounds also include an RNAi comprising a coding sequence and a non-coding sequence, in which the coding and non-coding sequences form an RNA duplex, and in which the non-coding sequence comprises a nucleotide sequence sufficiently complementary to the sequence. nucleotides of a HCBI5.173 polynucleic acid of the present invention.

Alternatively, the invention provides a vector that allows an antisense oligonucleotide of the invention to be transcribed, for example, in a mammalian host. Preferably, said vector is a useful vector for gene therapy. Preferred vectors useful for gene therapy are viral vectors, e.g. ex. adenovirus, adeno-associated virus, herpes simplex virus, vaccinia or, more preferably, an RNA virus such as a retrovirus. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of such retroviral vectors that can be used in the present invention are: Moloney murine leukemia virus (MoMuLV), Harvey's murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous sarcoma virus ( RSV). Most preferably, a non-human primate retroviral vector is employed, such as gibbon monkey leukemia virus (GaLV), which provides a broader host range compared to murine vectors. Because recombinant retroviruses are defective, assistance is required to produce infectious particles. Such assistance can be provided, for example, through the use of helper cell lines containing plasmids that encode all the structural genes of the retrovirus with the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those of skill in the art. Such vectors may additionally contain a gene encoding a selectable marker so that the transduced cells can be identified. Furthermore, retroviral vectors can be modified in such a way that they become target specific. This can be achieved, for example, by inserting a polynucleotide that encodes a sugar, a glycolipid or a protein, preferably an antibody. Additional procedures for generating target specific vectors are known to those of skill in the art. Other vectors and methods suitable for in vitro or in vivo gene therapy are described in the reference and are known to those of skill in the art; see, for example, WO 94/29469 or WO 97/00957. The HCBI polynucleotide sequences of the invention can also serve as a suitable vector per se, either composed solely of rearranged HCBI sequences or chimeric HCBI host cell DNA sequences. Furthermore, the nucleotide sequences of the invention can be used for the construction of artificial chromosomes.

To achieve expression only in the target organ, the DNA sequences for the transcription of the antisense oligonucleotides can be linked to a tissue-specific promoter and used for gene therapy. Such promoters are well known to those of skill in the art.

Within an oligonucleotide structure, phosphate groups are commonly related to the formation of the oligonucleotide's internucleoside backbone. The normal bond or backbone of RNA and DNA is a 3 'to 5' phosphodiester bond. Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or unnatural internucleoside linkages. Oligonucleotides that have modified backbones include those that retain a phosphorous atom in the backbone and those that do not have a phosphorous atom in the backbone. Modified oligonucleotide backbones that can result in greater stability are known to those skilled in the art, preferably said modification is a phosphorothioate bond.

A preferred oligonucleotide mimetic is an oligonucleotide mimetic which has been shown to have excellent hybridization properties, and is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and attached directly or indirectly to aza nitrogen atoms of the amide portion of the main chain.

Modified oligonucleotides can also contain one or more substituted or modified sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 'position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or 0-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be C1 to C10 alkyl or substituted or unsubstituted C2 to C10 alkenyl and alkynyl. A particularly preferred modified sugar residue is a 2'-O-methoxyethyl sugar residue.

The antisense oligonucleotides of the invention can also include nucleobase modifications or substitutions. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine etc., being preferred 5-methylcytosine substitutions, as these modifications have been shown to increase the stability of double-stranded nucleic acid.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, a thiocholesterol, an aliphatic chain, eg, dodecandiol or undecyl moieties, a phospholipid, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl radical, or an octadecylamine radical or a hexylaminocarbonyloxycholesterol radical.

The present invention also includes antisense compounds that are chimeric compounds. Antisense "chimeric" or "chimeras" compounds, in the context of the present invention, are antisense compounds, particularly oligonucleotides, containing two or more chemically distinct regions, each composed of at least one monomeric unit, that is, a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region in which the oligonucleotide is modified to confer on the oligonucleotide greater resistance to nuclease degradation, greater cell uptake, and / or greater binding affinity for the target nucleic acid. An additional region of the oligonucleotide can serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. By way of example, RNase H is a cellular endonuclease that clears the RNA strand of an RNA: DNA duplex. Activation of RNase H therefore results in cleavage of the target from RNA, greatly enhancing the efficiency of oligonucleotide gene expression inhibition. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. The chimeric antisense compounds of the invention can be formed as structures composed of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or void oligomers.

The present invention also relates to a pharmaceutical composition comprising an antibody or antisense oligonucleotide of the invention and a suitable excipient, diluent or carrier. Preferably, in a pharmaceutical composition, the compound as described above is combined with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" is intended to encompass any vehicle, which does not interfere with the efficacy of the biological activity of the active ingredient and which is not toxic to the host to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such vehicles can be formulated by conventional procedures and the active ingredient can be administered to the subject in an effective dose.

An "effective dose" refers to an amount of the active ingredient that is sufficient to prevent disease or to affect the course and severity of the disease, leading to reduction or remission of said disease. An "effective dose" useful for treating and / or preventing these diseases or disorders can be determined using procedures known to those of skill in the art.

Administration of suitable compositions can be effected in different ways, eg by intravenous, intraperitoneal, oral, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, the doses for any patient depend on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the type therapy, general health and other drugs that are administered simultaneously.

In a preferred embodiment of the present invention, the preventable / treatable disease is cancer, preferably colorectal, breast, prostate and pancreatic cancer, or a CNS disease, preferably Alzheimer's disease or multiple sclerosis (MS). The terms "cancer" and "CNS disease" also include the initial stages of these diseases.

Due to a compilation of global epidemiological data published by the inventor ( zur Hausen, 2012), evidence accumulated for putative infectious factors, which play a role in cancer, particularly in the colon, and possibly also other human cancers, transmitted from cattle to humans either by consumption of meat or dairy products. For these reasons, the inventors began testing 130 healthy cow sera for the presence of as yet unknown infectious agents. After isolating the five new DNAs shown in Fig. 1-5 in an initial series, the inventors began to search for the presence of their DNA in cells derived from cancer, in particular colon cancer. Simultaneously, materials from patients with multiple sclerosis were analyzed. Because multiple sclerosis represents a major human disease with often disastrous consequences for affected patients, the role of isolates according to the present invention in neurological disorders, specifically MS, has been intensively evaluated. The Results show that the sequences are only found in diseased patients, but, so far, they have not been found in healthy subjects. This applies to both cancer patients and those with CNS diseases.

The present invention also relates to a vaccine for immunizing a mammal against an HCBI infection, comprising at least one HCBI polypeptide or polynucleic acid as defined above, in a pharmaceutically acceptable carrier.

A "vaccine" is an immunogenic composition capable of eliciting protection against HCBI, either partial or complete. A vaccine can also be useful for treating an individual, in which case it is called a therapeutic vaccine.

The term "therapeutic" refers to a composition capable of treating an infection by HCBI or diseases related to this infection. The term "effective amount" refers to an amount of epitope-bearing polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to immunoreact otherwise detectable in their desired system (eg, immunoassay). Preferably, the effective amount is sufficient to effect the treatment, as defined above. The exact amount needed will vary depending on the application. For vaccine applications or for the generation of polyclonal antiserum / antibodies, for example, the effective amount may vary depending on the species, age and general condition of the individual, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. The effective amounts will be within a relatively large, non-critical range. An appropriate effective amount can be easily determined using only routine experimentation. Preferred ranges of proteins for prophylaxis of diseases caused by HCBI are 0.01 to 100 pg / dose, preferably 0.1 to 50 pg / dose. Several doses per individual may be required to achieve a sufficient immune response and subsequent protection against HCBI infection and HCBI-related disease, respectively.

Pharmaceutically acceptable vehicles include any vehicle that does not induce the production of antibodies harmful to the individual receiving the vaccine. Suitable carriers are typically large, slow metabolizing macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. Such vehicles are well known to those of skill in the art.

Preferred adjuvants to improve the efficacy of the composition include, but are not limited to: aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in US Pat. US No. 4,606,918, N -acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'- 2'-dipalmitoyl sn-glycero-3-hydroxyphosphoryloxy) ethylamine (MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL TDM CWS) in an emulsion 2% squalene / Tween 80. Any of the 3 components MPL, TDM or CWS can also be used alone or in combination 2 to 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1 can be used (Syntex). In addition, Freund's Complete Adjuvant (CFA) and Freund's Incomplete Adjuvant (IFA) can be used for non-human research purposes and applications.

Immunogenic compositions will normally contain pharmaceutically acceptable carriers, such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.

Usually, immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid vehicles can also be prepared prior to injection. The preparation can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect. Proteins can also be incorporated into Immune Stimulating Complexes together with saponins, eg Quil A (ISCOMS).

The immunogenic compositions used as vaccines comprise a "sufficient amount" or "an immunologically effective amount" of the proteins of the present invention, as well as any of the other components mentioned above, as necessary. "Immunologically effective amount" means that administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending on the health and physical condition of the individual to be treated, the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the evaluation of the medical situation by treating physician and other relevant factors. The amount is expected to be within a relatively wide range that can be determined through routine testing. Typically, the amount will range from 0.01 to 1000 pg / dose, more particularly 0.1-100 pg / dose.

Example 1

Material and Procedures

(A) Fractionation of bovine sera in density-sedimentation gradients with subsequent cloning

Bovine sera were separated in Optiprep (iodixanol) step gradients as previously described (Buck et al., 2004). Fractions were collected and DNA was extracted. Rolling circle amplification (from Villiers et al., 2011) was performed on each fraction prior to digestion with restriction enzymes BamH1 and EcoR1. The resulting fragments were cloned into pUC18 or pUC19 prior to sequencing.

(B) In vitro replication (from Villiers et al., 2011) of HCBI1.225 (Comparative example)

Linear full length HCBI1.225 was transfected into 293TT cells (Lipofectamine). The cultures were grown every 2-3 days. DNA and RNA were extracted from a fraction of the cells on passage. Input DNA was removed by Dpn1 digestion. HCBI1.225 DNA and RNA were expressed up to day 17 in cell culture.

Example 2

Characterization of the HCBI sequences

120 sera from healthy cows obtained from the "Veterinarinstitut der Universitat Leipzig" (Professor Muller) were analyzed and after viral particle purification, DNA extraction and "rolling circle" amplification were isolated (de Villiers et al., 2011 ) that possibly interact with circular viral DNA molecules that differ in size. The five DNA sequences and the open reading frames of these components showed a recognizable relationship with the sequences that have already been described for transmissible spongiform encephalopathies (TSE) for TSE diseases in sheep, cattle and humans (Manuelidis, 2011 ). These agents were not further characterized and were designated "Sphinx viruses". Since the DNA sequences (and open reading frames) obtained in the present invention from healthy cows show homologies with the corresponding sequences described by Manuelidis, but are not identical, the designation HCBI (Healthy Cattle Blood jsolate) is proposed.

In this context, CNS diseases (eg multiple sclerosis, Alzheimer's disease) are also very interesting since the similar sequences described by Manuelidis are mainly found in the CNS.

The "Sphinx" sequences (Manuelidis, 2011) show high homologies with the plasmid sequences of the Acinetobacter bacterium (Vallenet et al., 2008; Longkummer et al., 2013). The sequences obtained in the present invention also show surprising homologies with the sequences of the corresponding plasmids. Therefore, at first glance, it is tempting to speculate that the results are due to contamination of the samples with Acinetobacter viruses. Although a plethora of Acinetobacter plasmids have in the meantime been sequenced, none of these sequences correspond to the DNA sequences of the present invention that were obtained from three different lots of sera. Since the isolation of these DNAs was initially achieved by purification of the viruses through density gradient centrifugation, the apparently common presence of these viruses in the blood of cow sera is not necessarily attributable to the viral contamination derived. of bacteria. Furthermore, the presence of DNA sequences of these viruses in human cancer cells is incompatible with this interpretation.

Interestingly, a group of scientists in the United Kingdom published serological data over a period of years pointing to increased selective formation of antibodies against Acinetobacter proteins, but not against other bacterial antigens obtained from patients suffering from multiple sclerosis (see article review: Ebringer et al., 2012). These results could not be confirmed by Chapman's group (Chapman et al., 2005). However, it should be emphasized that Chapman's group used a different strain of Acinetobacter (Acinetobacter calcoaceticus). Unambiguous results were obtained by Ebringer's group for three Acinetobacter strains ( Acinetobacter Iwoffii, A. radioesistens and a specific isolate, A. 11171). However, the results obtained for A. junii 17908 were less impressive and the significant reactivity was hardly detectable (Hughes et al., 2001). These results suggest that we are dealing with strain-specific reactivities in which this serological reactivity is due to strain-specific plasmids showing homologies with the DNA sequences obtained in the present invention.

References

Buck CB, Pastrana DV, Lowy DR, Schiller JT. Efficient intracellular assembly of papillomaviral vectors. J. Virol.

2004; 78: 751-757.

Chapman MD, Hughes LE, Wilson CD, Namnyak S, Thompson EJ, Giovannoni G. No evidence for production of intrathecal immunoglobulin G against Acinetobacter or Pseudomonas in multiple sclerosis. Eur Neurol. 2005; 53 (1): 27-31.

by Villiers EM, Borkosky SS, Kimmel R, Gunst K and Fei JW. (2011) The diversity of Torque teno viruses: In vitro replication leads to the formation of additional replication-competent subviral Molecules. J Virol 2011; 85 (14): 7284-7295

Ebringer A, Hughes LE, Rashid T, Wilson C. Acinetobacter Immune Responses in Multiple Sclerosis:

Claims (11)

1. An HCBI polynucleic acid comprising: (a) a nucleotide sequence depicted in Figure 5 (SEQ ID. NO: 72); (b) a nucleotide sequence that is at least 90% identical to a nucleotide sequence of (a); (c) a nucleotide sequence that is complementary to a nucleotide sequence of (a) or (b); or (d) a nucleotide sequence that is redundant as a result of the degeneracy of the genetic code compared to any of the nucleotide sequences given above. 2. An oligonucleotide primer comprising part of a HCBI polynucleic acid of claim 1, said primer being capable of acting as a primer to specifically sequence or specifically amplify the nucleic acid of a given HCBI isolate containing a nucleotide sequence of claim 1. 3. An oligonucleotide probe comprising part of a HCBI polynucleic acid of claim 1, said probe being capable of acting as a hybridization probe for the specific detection of the nucleic acid of a given HCBI isolate containing a nucleotide sequence of claim 1. 4. An expression vector comprising an HCBI polynucleic acid of claim 1 operably linked to prokaryotic, eukaryotic or viral transcription and translation control elements. 5. A host cell transformed with an expression vector according to claim 4. 6. A polypeptide that is encoded by an HCBI polynucleic acid of claim 1. 7. An antibody or antigen-binding fragment thereof that specifically binds a polypeptide of claim 6. A method for the detection of a HCBI polynucleic acid according to claim 1 in a biological sample, comprising: (a) optionally extracting the polynucleic acid from the sample, (b) amplifying the polynucleic acid as described described above with at least one primer according to claim 2, optionally a labeled primer, and (c) detecting the amplified polynucleic acid. A method for the detection of an HCBI polynucleic acid according to claim 1 in a biological sample, comprising: (a) optionally extracting the polynucleic acid from the sample, (b) hybridizing the polynucleic acid as described described above with at least one probe according to claim 3, optionally a labeled probe, and (c) detecting the hybridized polynucleic acid. A method for detecting a polypeptide of claim 6 or an antibody of claim 7 present in a biological sample, comprising: (a) contacting the biological sample to detect the presence of said polypeptide or antibody as defined above , and (b) detecting the immune complex formed between said antibody and said polypeptide. 11. An antisense oligonucleotide that reduces or inhibits the expression of an HCBI polynucleic acid of claim 1 or a vector containing said antisense oligonucleotide.